The present disclosure relates to non-immunosuppressive cyclosporin analogues which bind to cyclophilins, which are cyclophilin inhibitors, in particular to their pharmaceutical use in the treatment of infection with Coronaviurs (CoV).
Coronaviruses cause severe diseases of the respiratory and gastrointestinal tract and the central nervous system in animals (Perlman, S., Netland, J., 2009. Coronaviruses post-SARS: update on replication and pathogenesis. Nat. Rev. Microbiol 7(6), 439-450). The infection of humans with HCoV-OC43 and HCoV-229E are known since the mid sixties to be associated with respiratory tract i.e. common cold-like diseases. SARS-CoV (Severe Acute Respiratory Syndrome-Corona Virus) is a highly aggressive human agent, causing the lung disease SARS, with often fatal outcome (Drosten, C., Gunther, S., Preiser, W., van der, W. S., Brodt, H. R., Becker, S., Rabenau, H., Panning, M., Kolesnikova, L., Fouchier, R. A., Berger, A., Burguiere, A. M., Cinatl, J., Eickmann, M., Escriou, N., Grywna, K., Kramme, S., Manuguerra, J. C., Muller, S., Rickerts, V., Sturmer, M., Vieth, S., Klenk, H. D., Osterhaus, A. D., Schmitz, H., Doerr, H. W., 2003. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N. Engl. J. Med. 348(20), 1967-1976). This virus appeared as an epidemic in 2003 after it had crossed the species barrier from bats to civet cats and humans demonstrating the potential of coronaviruses to cause high morbidity and mortality in humans (Lau, S. K., Woo, P. C., Li, K. S., Huang, Y., Tsoi, H. W., Wong, B. H., Wong, S. S., Leung, S. Y., Chan, K. H., Yuen, K. Y., 2005. Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats. Proc Natl Acad Sci USA 102(39), 14040-14045; Li, W., Shi, Z., Yu, M., Ren, W., Smith, C., Epstein, J. H., Wang, H., Crameri, G., Hu, Z., Zhang, H., Zhang, J., McEachern, J., Field, H., Daszak, P., Eaton, B. T., Zhang, S., Wang, L. F., 2005. Bats are natural reservoirs of SARS-like coronaviruses. Science 310(5748), 676-679). As no treatment was available, the epidemic could eventually be controlled by highly effective traditional public health measures of quarantine and case isolation. The strains HCoV-NL63 and HCoV-HKU1 were discovered in 2004 and 2005, respectively (van der Hoek, L., Pyrc, K., Jebbink, M. F., Vermeulen-Oost, W., Berkhout, R. J., Wolthers, K. C., Wertheim-van Dillen, P. M., Kaandorp, J., Spaargaren, J., Berkhout, B., 2004. Identification of a new human coronavirus. Nat Med 10(4), 368-373; Woo, P. C., Lau, S. K., Chu, C. M., Chan, K. H., Tsoi, H. W., Huang, Y., Wong, B. H., Poon, R. W., Cai, J. J., Luk, W. K., Poon, L. L., Wong, S. S., Guan, Y., Peiris, J. S., Yuen, K. Y., 2005. Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia. Journal of virology 79(2), 884-895). They cause more severe lower respiratory tract infections like bronchiolitis and pneumonia especially in young children, immunocompromised patients and the elderly (van der Hoek, L., 2007. Human coronaviruses: what do they cause? Antivir Ther 12(4 Pt B), 651-658). In 2012, a new human CoV MERS (Middle East Respiratory Syndrome virus, previously called “EMC”) emerged from the Middle East with clinical outcomes such as renal failure and acute pneumonia, similar to those of SARS-CoV but with an even higher mortality rate of about 50% (de Groot, R. J., Baker, S. C., Baric, R. S., Brown, C. S., Drosten, C., Enjuanes, L., Fouchier, R. A., Galiano, M., Gorbalenya, A. E., Memish, Z., Perlman, S., Poon, L. L., Snijder, E. J., Stephens, G. M., Woo, P. C., Zaki, A. M., Zambon, M., Ziebuhr, J., 2013. Middle East Respiratory Syndrome Coronavirus (MERS-CoV); Announcement of the Coronavirus Study Group. Journal of virology; van Boheemen, S., de Graaf, M., Lauber, C., Bestebroer, T. M., Raj, V. S., Zaki, A. M., Osterhaus, A. D., Haagmans, B. L., Gorbalenya, A. E., Snijder, E. J., Fouchier, R. A., 2012. Genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans. MBio 3(6); Zaki, A. M., van Boheemen, S., Bestebroer, T. M., Osterhaus, A. D., Fouchier, R. A., 2012. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med 367(19), 1814-1820).
Human coronaviruses cause approximately 10-15% of all upper and lower respiratory tract infections. They account for significant hospitalizations of children under 18 years of age, the elderly and immunocompromised individuals. According to a number of international studies 5-10% of the acute respiratory diseases are caused by HCoV-NL63 [for review see Abdul-Rasool, S., Fielding, B. C., 2010. Understanding Human Coroonavirus HCoV-NL63. The Open Virology Journal 4, 76-84]. These numbers are probably a great underestimation since during diagnostic screening for respiratory viruses tests for HCoV's are frequently not included. An important aspect HCoV-NL63 infection is the co-infection with other human coronaviruses, influenza A, respiratory syncytial virus (RSV), parainfluenza virus human metapneumovirus (Abdul-Rasool, S., Fielding, B. C., 2010. Understanding Human Coroonavirus HCoV-NL63. The Open Virology Journal 4, 76-84). In children they are associated with acute respiratory tract illness, pneumonia and Croup leading in many cases to hospitalization. In a recent epidemiological study out of 1471 hospitalized children (<2 years) 207 (14%) were HCoV-positive (Dijkman, R., Jebbink, M. F., Gaunt, E., Rossen, J. W., Templeton, K. E., Kuijpers, T. W., van der Hoek, L., 2012. The dominance of human coronavirus OC43 and NL63 infections in infants. Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 53(2), 135-139). Infection frequencies in children with mild symptoms and in hospitalized children occurred in the order HCoV-OC43>HCoV-NL63>HCoV-HKU1>HCoV-229E. In a large-scale survey on 11,661 diagnostic respiratory samples collected in Edinburgh, UK, between 2006 and 2009, 267 (2.30%) were positive for at least one coronavirus accounting for 8.15% of all virus detections (Gaunt, E. R., Hardie, A., Claas, E. C., Simmonds, P., Templeton, K. E., 2010. Epidemiology and clinical presentations of the four human coronaviruses 229E, HKU1, NL63, and OC43 detected over 3 years using a novel multiplex real-time PCR method. Journal of clinical microbiology 48(8), 2940-2947). 11 to 41% of coronaviruses detected were present in samples tested positive for other respiratory viruses (e.g. RSV).
Although inhibitors of coronavirus enzymes (reviewed by (Tong, T. R., 2009a. Therapies for coronaviruses. Part 2: Inhibitors of intracellular life cycle. Expert. Opin. Ther. Pat 19(4), 415-431; Tong, T. R., 2009b. Therapies for coronaviruses. Part I of II—viral entry inhibitors. Expert. Opin. Ther. Pat 19(3), 357-367) and compounds inhibiting in vitro replication have been described (Kono, M., Tatsumi, K., Imai, A. M., Saito, K., Kuriyama, T., Shirasawa, H., 2008. Inhibition of human coronavirus 229E infection in human epithelial lung cells (L132) by chloroquine: involvement of p38 MAPK and ERK. Antiviral research 77(2), 150-152; to Velthuis, A. J., van den Worm, S. H., Sims, A. C., Baric, R. S., Snijder, E. J., van Hemert, M. J., 2010. Zn(2+) inhibits coronavirus and arterivirus RNA polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture. PLoS Pathog 6(11), e1001176; Vincent, M. J., Bergeron, E., Benjannet, S., Erickson, B. R., Rollin, P. E., Ksiazek, T. G., Seidah, N. G., Nichol, S. T., 2005. Chloroquine is a potent inhibitor of SARS coronavirus infection and spread. Virol J 2, 69), clinically licensed antivirals for coronavirus infection are absent. Coronaviruses represent the largest group of single-stranded RNA viruses with plus strand orientation. Thus they are prone to evolutionary change due to lack of proof reading activity of its polymerases provoking the development of resistance mutations in the presence of inhibitors of viral proteins. Virus replication depends on a variety of host factors (de Haan, C. A., Rottier, P. J., 2006. Hosting the severe acute respiratory syndrome coronavirus: specific cell factors required for infection. Cellular microbiology 8(8), 1211-1218; Vogels, M. W., van Balkom, B. W., Kaloyanova, D. V., Batenburg, J. J., Heck, A. J., Helms, J. B., Rottier, P. J., de Haan, C. A., 2011. Identification of host factors involved in coronavirus replication by quantitative proteomics analysis. Proteomics 11(1), 64-80; Wang, R. Y., Li, K., 2012. Host factors in the replication of positive-strand RNA viruses. Chang Gung medical journal 35(2), 111-124) which represent potential antiviral targets. These might be more preferable targets than viral proteins as development of resistance is much less likely.
In a recent study we performed a genome-wide SARS-CoV yeast-two-hybrid interaction screen with human cDNA libraries identifying human immunophilins (including cyclophilins [Cyps] and FK506-binding proteins [FKBPs] as interaction partners of CoV non-structural protein 1 [Nsp1](Pfefferle, S., Schopf, J., Kogl, M., Friedel, C. C., Muller, M. A., Carbajo-Lozoya, J., Stellberger, T., von Dall'Armi, E., Herzog, P., Kallies, S., Niemeyer, D., Ditt, V., Kuri, T., Zust, R., Pumpor, K., Hilgenfeld, R., Schwarz, F., Zimmer, R., Steffen, I., Weber, F., Thiel, V., Herrler, G., Thiel, H. J., Schwegmann-Wessels, C., Pohlmann, S., Haas, J., Drosten, C., von Brunn, A., 2011. The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors. PLoS Pathog 7(10), e1002331). A pronounced feature of most mammalian cyclophilins is their ability to bind the immunosuppressive drug cyclosporine A (CsA). We showed that the drug acts as a replication inhibitor of a number of human (SARS-CoV, HCoV-NL63 and HCoV-229E) and animal coronaviruses (Feline CoV [serotypes I and II], porcine transmissible gastroenteritis virus (TGEV), and avian infectious bronchitis virus [IBV]) suggesting host cyclophilins as targets for pan-coronavirus inhibition (Pfefferle, S., Schopf, J., Kogl, M., Friedel, C. C., Muller, M. A., Carbajo-Lozoya, J., Stellberger, T., von Dall'Armi, E., Herzog, P., Kallies, S., Niemeyer, D., Ditt, V., Kuri, T., Zust, R., Pumpor, K., Hilgenfeld, R., Schwarz, F., Zimmer, R., Steffen, I., Weber, F., Thiel, V., Herrler, G., Thiel, H. J., Schwegmann-Wessels, C., Pohlmann, S., Haas, J., Drosten, C., von Brunn, A., 2011. The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors. PLoS Pathog 7(10), e1002331). Inhibition of SARS-CoV, HCoV-229E and in addition of MHV was subsequently also confirmed by de Wilde et al. (de Wilde, A. H., Zevenhoven-Dobbe, J. C., van der Meer, Y., Thiel, V., Narayanan, K., Makino, S., Snijder, E. J., van Hemert, M. J., 2011. Cyclosporin A inhibits the replication of diverse coronaviruses. The Journal of general virology 92(Pt 11), 2542-2548). Inhibition of feline CoV replication was also found by Tanaka et al. (Tanaka, Y., Sato, Y., Osawa, S., Inoue, M., Tanaka, S., Sasaki, T., 2012. Suppression of feline coronavirus replication in vitro by cyclosporin A. Veterinary research 43(1), 41). Similarly, we showed that FK506 inhibits the replication of SARS-CoV, HCoV-NL63 and HCoV-229E and the dependence of HCoV-NL63 on FKBP1A/B Carbajo-Lozoya, J., Muller, M. A., Kallies, S., Thiel, V., Drosten, C., von Brunn, A., 2012. Replication of human coronaviruses SARS-CoV, HCoV-NL63 and HCoV-229E is inhibited by the drug FK506. Virus Res 165(1), 112-117).
Cyclophilins and FKBPs represent large, independent families of peptidyl-prolyl cis/trans isomerases (PPIases, EC number 5.2.1.8) thus exerting important functions on folding, maturation and trafficking of proteins within the eukaryotic cell (Blackburn, E. A., Walkinshaw, M. D., 2011. Targeting FKBP isoforms with small-molecule ligands. Current opinion in pharmacology 11(4), 365-371; Davis, T. L., Walker, J. R., Campagna-Slater, V., Finerty, P. J., Paramanathan, R., Bernstein, G., MacKenzie, F., Tempel, W., Ouyang, H., Lee, W. H., Eisenmesser, E. Z., Dhe-Paganon, S., 2010. Structural and biochemical characterization of the human cyclophilin family of peptidyl-prolyl isomerases. PLoS Biol 8(7), e1000439). Both CsA and FK-506 act as tight-binding, reversible and competitive inhibitors of the active site of these enzymes (Fischer, G., Wittmann-Liebold, B., Lang, K., Kiefhaber, T., Schmid, F. X., 1989. Cyclophilin and peptidyl-prolyl cis-trans isomerase are probably identical proteins. Nature 337(6206), 476-478). Physical interaction of cyclophilins with viral proteins, and thus replication sensitivity to CsA have been shown for several viruses, e.g. the capsid proteins of HIV-1 (Strebel, K., Luban, J., Jeang, K. T., 2009. Human cellular restriction factors that target HIV-1 replication. BMC Med 7, 48; Ylinen, L. M., Schaller, T., Price, A., Fletcher, A. J., Noursadeghi, M., James, L. C., Towers, G. J., 2009. Cyclophilin A levels dictate infection efficiency of human immunodeficiency virus type 1 capsid escape mutants A92E and G94D. Journal of virology 83(4), 2044-2047) and HPV types 16 (Bienkowska-Haba, M., Patel, H. D., Sapp, M., 2009. Target cell cyclophilins facilitate human papillomavirus type 16 infection. PLoS Pathog 5(7), e1000524), the N protein of Vesicular stomatitis Virus (Bose, S., Mathur, M., Bates, P., Joshi, N., Banerjee, A. K., 2003. Requirement for cyclophilin A for the replication of vesicular stomatitis virus New Jersey serotype. The Journal of general virology 84(Pt 7), 1687-1699), the NS5a of HCV (Fernandes, F., Ansari, I. U., Striker, R., 2010. Cyclosporine inhibits a direct interaction between cyclophilins and hepatitis C NS5A. PloS one 5(3), e9815; Fischer, G., Wittmann-Liebold, B., Lang, K., Kiefhaber, T., Schmid, F. X., 1989. Cyclophilin and peptidyl-prolyl cis-trans isomerase are probably identical proteins. Nature 337(6206), 476-478), the NS4A protein of the mosquito-borne Japanese encephalitis virus (Kambara, H., Tani, H., Mori, Y., Abe, T., Katoh, H., Fukuhara, T., Taguwa, S., Moriishi, K., Matsuura, Y., 2011. Involvement of cyclophilin B in the replication of Japanese encephalitis virus. Virology 412(1), 211-219), the NS5 protein of West Nile virus (Qing, M., Yang, F., Zhang, B., Zou, G., Robida, J. M., Yuan, Z., Tang, H., Shi, P. Y., 2009. Cyclosporine inhibits flavivirus replication through blocking the interaction between host cyclophilins and viral NS5 protein. Antimicrobial agents and chemotherapy 53(8), 3226-3235) and the M1 protein of influenza A virus (Liu, X., Sun, L., Yu, M., Wang, Z., Xu, C., Xue, Q., Zhang, K., Ye, X., Kitamura, Y., Liu, W., 2009. Cyclophilin A interacts with influenza A virus M1 protein and impairs the early stage of the viral replication. Cellular microbiology 11(5), 730-741). The most prominent cyclophilins thought to be involved are CypA and CypB. PPIase-independent activities of CsA and FK506 exerted by gain-of-function, result from the binary complexes formed by binding of the drugs to Cyps and FKBPs, respectively. Based on the inhibition of the protein phosphatase activity of calcineurin, these complexes block the cellular calcineurin (CaN)/NFAT pathway thereby interfering with T-cell activation and 11-2 production. Chemically changed derivatives covering specific side-chain modifications, the so-called non-immunosuppressive cyclosporine- or FK506, analogues, can discriminate between alternative signalling pathways either based on PPIase- or CaN-inhibiting functions.
Identifying the interaction of the SARS-CoV Nsp1 protein with Cyps and FKBPs, and the sensitivity of CoV replication to both drugs, CsA and FK506, CsA was suggested as a potential pan-CoV inhibitor (Pfefferle, S., Schopf, J., Kogl, M., Friedel, C. C., Muller, M. A., Carbajo-Lozoya, J., Stellberger, T., von Dall'Armi, E., Herzog, P., Kallies, S., Niemeyer, D., Ditt, V., Kuri, T., Zust, R., Pumpor, K., Hilgenfeld, R., Schwarz, F., Zimmer, R., Steffen, I., Weber, F., Thiel, V., Herrler, G., Thiel, H. J., Schwegmann-Wessels, C., Pohlmann, S., Haas, J., Drosten, C., von Brunn, A., 2011. The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors. PLoS Pathog 7(10), e1002331). Here it is demonstrated that, by using Alisporivir, NIM811 and a series of newly synthesized CsA and FK506 derivatives, inhibition of HCoV-NL63 replication independent of the immunosuppressive character of the compounds. It is further shown that CypA but not CypB is required for virus replication.
The cyclosporins and the non-immunosuppressive analogues comprise a class of structurally distinctive, cyclic, poly-N-methylated undecapeptides, commonly possessing pharmacological, in particular immunosuppressive, or anti-inflammatory activity. The first of the cyclosporins to be isolated was the naturally occurring fungal metabolite Ciclosporin or Cyclosporine, also known as cyclosporin A (CsA). Cyclosporins which bind strongly to cyclophilin but are not immunosuppressive have been identified. PCT/EP 2004/009804, WO 2005/021028, or WO 2006/071619 (which are incorporated by reference herein in their entirety) disclose non-immunosuppressive cyclosporins which bind to cyclophilin and have also been found to have an inhibitory effect on Hepatitis C virus (HCV). WO 2006/038088, incorporated herein by reference in its entirety, describes methods and compositions for the use of alisporivir in the treatment of HCV. Alisporivir (DEB025 or Debio-025) is a cyclophilin (Cyp) inhibitor and its mode of action as an anti-HCV agent is via inhibition of host proteins, in particular of cyclophilin A, that are directly involved in HCV replication.
Therefore it is an object of the present disclosure to provide new methods for the treatment of patients with Coronavirus infection alone or patients infected co-infected with an additional Coronavirus.
Surprisingly it has been found that non-immunosupressive cyclophilin inhibitors, in particular alisporivir and NIM811, have antiviral properties against Coronavirus that can be used effectively in the treatment of CoV infections. In particular, it has been found that the non-immunosuppressive cyclophilin inhibitors alisporivir and NIM811 inhibit CoV replication independent of the immunosuppressive character of the compounds. Accordingly, the present invention provides new anti-CoV treatments using alisporivir and NIM811.
Furthermore, the present disclosure provides methods for the treatment of CoV and CoV-co-infections comprising administering an effective amount of a non-immunosuppressive cyclophilin inhibitor, in particular alisporivir and/or NIM811, either alone or in combination with another antiviral agent such as ribavirin.